LDLR is used as a cell entry receptor by multiple alphaviruses

Alphaviruses are arboviruses transmitted by mosquitoes and are pathogenic to humans and livestock, causing a substantial public health burden. So far, several receptors have been identified for alphavirus entry; however, they cannot explain the broad host range and tissue tropism of certain alphaviruses, such as Getah virus (GETV), indicating the existence of additional receptors. Here we identify the evolutionarily conserved low-density lipoprotein receptor (LDLR) as a new cell entry factor for GETV, Semliki Forest virus (SFV), Ross River virus (RRV) and Bebaru virus (BEBV). Ectopic expression of LDLR facilitates cellular binding and internalization of GETV, which is mediated by the interaction between the E2-E1 spike of GETV and the ligand-binding domain (LBD) of LDLR. Antibodies against LBD block GETV infection in cultured cells. In addition, the GST-LBD fusion protein inhibits GETV infection both in vitro and in vivo. Notably, we identify the key amino acids in LDLR-LBD that played a crucial role in viral entry; specific mutations in the CR4 and CR5 domain of LDLR-LBD reduce viral entry to cells by more than 20-fold. These findings suggest that targeting the LDLR-LBD could be a potential strategy for the development of antivirals against multiple alphaviruses.

1.The data in Figure 1d-k were generated with lentivirus pseudotypes.These lentivirus pseudotypes are unlikely to contain properly assembled E2/E1 protomers (with icosahedral assembly).So, while this experiment is well controlled (e.g., expected phenotypes are seen with Mxra8 overexpression with chikungunya virus pseudotypes), it remains critical to also use a different system that faithfully recapitulates alphavirus spike protein organization to make a claim on receptor use for SFV, BEBV, and RRV (e.g., chimeric Sindbis viruses, which are the standard in the field, or authentic alphaviruses).
For example, a separate study found only low levels of entry of alphavirus replicon-based SFV reporter virus particles with cells overexpressing LDLR; but a much more significant effect was seen with VLDLR overexpression (PMID: 34929721).Structural analysis of VLDLR bound to SFV virus-like particles also suggests that there may be important requirements for receptor assembly near icosahedral axes of the E2-E1 spike proteins (PMID: 37098345).It is thus possible that sites that might be exposed for receptor binding by LDLR on SFV-lentivirus pseudotypes might not be exposed on properly assembled E2-E1 spikes in the context of native or native-like virions.
2. Critical assays for SFV, BEBV, and RRV are missing to conclude that LDLR is a receptor for these viruses.These assays include: (a) Showing that LDLR binds to the E2-E1 proteins of these viruses through immunoprecipitation experiments, ELISAs, or BLI experiments.(b) Assays showing that LDLR expression results in binding to cells and virus internalization.(c) As noted above, assays with authentic viruses are missing.
3. VLDLR, a known alphavirus receptor that is very closely related to LDLR and is also a receptor for multiple alphaviruses including SFV, was not tested anywhere in the manuscript.VLDLR (and ApoER2) are two canonical members of the LDLR family that have similar structural organization and sequence homology in their ligand-binding domains to LDLR (while LDLRAD3, which the authors tested, is quite different).While the manuscript centers on LDLR, interest would be significantly increased if the authors demonstrated that LDLR but not VLDLR or ApoER2 are cellular receptors for GETV.This finding would have important implications to the general understanding of the evolution of alphavirus receptor specificities.
In a related question, were VLDLR and ApoER2 on the list of the 150 membrane proteins tested in their screen?The list of membrane proteins should be provided in the supplemental materials for completeness.
4. Biolayer interferometry assays in Figure 3i.The authors dipped sensor tips that were coated with receptors into solution containing VLPs, and report measuring an affinity of 263 nM.There would be substantial avidity in the system, which would falsely decrease the off rate.Any KD determination would therefore be uninterpretable.For affinity measurements, the virus would have to be immobilized on the sensor tip and the receptor kept in solution to minimize avidity.4 e-h rely on mouse anti-LDLR serum, which may have some cross-reactivity with other LDLR family members (e.g., with VLDLR) and confound the results of this assay.Could the authors comment on how this antibody was validated, and is it possible that it may cross-react with VLDLR, LRP1, etc.? 6.For the confocal microscopy studies (Figure 2k), how many images were used to quantify binding?Could additional images be provided in the supplement? 7. Abstract: "Although several receptors have been identified for alphavirus entry, they could not explain broad host range and multi-tissue tropism of alphaviruses, indicating the existence of additional receptors."This reviewer does not agree with this statement.Another, very closely related LDLR-related family receptor, VLDLR, as already been described as a conserved receptor for SFV, and orthologs from vertebrates and invertebrates are functional receptors.Would recommend rephrasing to say that some of the previously identified receptors cannot explain the broad host range and tissue tropism of certain alphaviruses.

Minor:
Lines 70-71: "However, ApoER2 and VLDLR are almost exclusively expressed in the central nervous system" While ApoER2 expression is mostly limited to the brain, VLDLR is more widely expressed, with expression in the heart, muscle, lung, kidneys, and adipose tissue.See reference PMID: 21554715.

Reviewer #2 (Remarks to the Author):
This manuscript by Zhai et al. describes a test of 150 membrane proteins for roles in promoting infection by Getah Virus.GETV is an alphavirus that is an important pathogen of pigs, and knowledge of its receptor is important in understanding infection and pathogenesis.The paper reports that LDLR is a receptor for GETV, and also for SFV, RRV, and BEBV.There have been in the last few years a number of very strong papers on alphavirus receptors, including both functional and structural studies: MXRA8 for CHIKV, LDLRAD3 for VEEV, VLDLR and ApoER2 for SFV and EEEV.Given these extensive studies, the bar to prove the role of a new alphavirus receptor is high.The current paper represents a considerable amount of work in testing viruses, testing various receptor constructs, mapping binding sites, and performing infection/blocking experiments in mice.However, there are serious concerns about the constructs and some other reagents used in the paper, and these issues need to be addressed thoroughly before the reader can be convinced of the importance of LDLR as a receptor for GETV and also for SFV, RRV, and BEBV.
Major points: 1.The paper suffers from a serious deficiency of careful documentation of the various constructs used.The library of 150 membrane proteins are tagged with eGFP-there is no documentation of whether the tag is on the N-or C-terminus, and whether it affects delivery of the protein to the membrane.More importantly, for the N-tagged LDLR constructs, there is no information on whether the FLAG tag is placed after the signal sequence, and if not, how the protein would be inserted into the ER.The studies lean heavily on the use of a soluble LDL LBD-GST protein, which is used to block infection in cell culture and in mouse studies, and to produce an antibody that is used in cell blocking experiments.This LBD-GST protein is produced in bacteria.There is no documentation that this heavily disulfide-bonded protein is folded correctly, or references to published work that might support this.Might this explain why the western blot and flow cytometry studies use commercial Abs to LDLR or FLAG, rather than the polyclonal antibody they generated?2. It is critical to confirm that the various LDLR mutants are expressed on the cell surface.This is addressed by flow cytometry and receptor staining.The receptors are described to be N-terminally tagged i.e. the FLAG tag should be localised extracellularly.However, the flow cytometry methods state that cells were permeabilised to detect the FLAG tag.From this they then conclude the receptor cell surface levels.This is not accurate as permeabilisation will allow the entire receptor population to be labelled (i.e both cytoplasmic and PM localised).Without cell surface confirmation of all receptor constructs, the data are not conclusive.(The methods also read like cells are fixed prior to trypsinisation, which would prevent the cells from coming off the plate for the flow analysis).3. The endocytosis assays in Fig. 2 are not convincing.There is no stripping step to remove bound virus after uptake (2J).The fluorescence figure in 2K shows binding of virus, but what is needed in addition is to demonstrate that the receptor promotes uptake (e.g., this was shown very convincingly in the Clark et al paper).Such experiments are critical to prove that LDLR is actually acting as a receptor.The discussion of the role of receptor in virus endocytosis should cite the highly relevant paper by Feng et al, J. Virol 2023, and also the receptor studies (MXRA8, LDLRAD3) that examined the role of the TM and cytoplasmic tail.4. Another key point is to demonstrate that GETV p62-E1 binds directly to the LDLR, ruling out other proteins acting as intermediaries.However, the p62-E1 construct that is produced in 293 cells was not purified for these studies, but a cell lysate prep was used.While the result may be correct, it would be much more convincing to demonstrate this with purified proteins.The GETV VLP studies look more convincing, but the authors should consider whether associated LDL molecules might explain the interaction (as was considered in the Clark et al paper).5.The sequence comparisons in extended data Fig. 8 are interesting and used to identify candidate sites of interaction (see also Fig. 6).However, these comparisons would be much stronger if the permissive CR 4 and CR5 domains were compared to the sequences of the nonpermissive CR 1, 2 etc.In ED Minor points: 1. Please briefly describe the construction of the rGETV-eGFP reporter virus, which does not seem to be in ref. 33.Is this a cytoplasmic reporter?If the construct is similar to that of rGETV-mCherry, is there a problem with the cysteines in eGFP?Does the virus grow efficiently?2. In the studies of the KO cells in extended data Fig. 4, why are there not results for both alleles of the various proteins?It would be quite surprising if both alleles had the same CRISPR-edited sequence for all of the KO.Please also explain if these are cell clones or populations.3. Throughout the paper, labeling of the constructs should accurately indicate the position of the tag.For example, the LDLR constructs state that they have N-terminal FLAG tags, but are designated LDLR-FLAG.This is confusing and not the usual convention.4. Line 66. Please adjust text to reflect that the mode of interaction (ie structural analysis) has also been determined for MXRA8 and LDLRAD3. 5. Line 71 is inaccurate.Both NCBI and Human protein atlas databases report very wide tissue expression of at least human VLDLR.6. Please adjust text in introduction to reflect that the Clark et al paper tested VLDLR from many species including mosquito.In this study, the authors identified LDLR as a crucial receptor involved in entry of several viruses belonging to the Alphavirus genus into target cells, whereby Getah virus was the main focus of the study due to biological safety reasons.The overall experimental strategy was well-designed.However, there is major concern if LDLR is indeed the crucial receptor for these viruses without validation experiments with in-vivo knockout model of LDLR.In-vivo model of LDLR knockout are well established and has been utilized for diseases such as hypercholesterolemia.
1) The authors have constructed an expression plasmid library for 150 mouse membrane proteins, in order to identify the specific protein(s) which positively affect alphavirus infections.What are the selection criteria of these proteins, have these proteins been shown in the past to be associated with the infection capacity of other viruses?
2) It will be useful for the authors to provide a list of the tested proteins including those which were negative hits in this study, as it can serve as an important source of information for future works by the readers.
3) Figure 2j: Can the authors provide a clear description on the y-axis of the graph?Is the y-axis presented as relative ratio of internalization:binding data?The error bars are too huge in my opinion to deduce that there is an effect on the virus internalization, while there are some data points which showed similar levels of vRNA as the WT. 4) In Figure 2t, the authors showed that JEV is among the virus which are not affected by LDLR deficiency in the host cells.However, this is in contrast to a previous finding (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238074/) which mechanistically demonstrated that LDLR is an important host factor required for JEV entry.The authors will need to address this contrasting data.5) Although the mouse sera used in this study was collected from mice subcutaneously injected with GST-LBD, how did the author confirm the presence of anti-LDLR antibodies in the sera generated from their mouse model?Was there any neutralization test being performed using the generated sera?The authors need to convincingly show that the inhibition observed on the rGETV-EGFP infection within the different cell lines tested are indeed due to the presence of anti-LDLR antibodies.
6) It is not convincing with the rather low levels of infection efficiency by the different viruses generated by the authors in Figure 1, it is perplexing with the extremely low levels of EGFP+ cells illustrated in Figures 3d, 6b and 6g, within the WT or empty vector control groups, which are rather contrasting and not even close to the initial data in Figure 1d showed an approximately 10% infection by GETV.Another concern from this point is that the rather low infection levels of the WT group might result in a biased over-interpretation of the efficiency of LDLR expression in the rest of the experiments in this study.7) Validation experiments of virus infectivity and pathogenesis with in-vivo knockout model of LDLR is essential.7) Figure 7b and c: It is inconclusive for the authors to deduce that the CR4 region are also essential for promoting GETV infection as there were still statistically significant upregulation of the EGFP+ cells and viral titers in the absence of CR4.8) Line 223: Was the promoting effect in the absence of CR4 statistically compared to CR5?It is inaccurate for the authors to state that "deletion of CR4 statistically reduced the promoting effect of LDLR expression", as the statistics performed were on the upregulation of EGFP+ cells and viral titers relative to the WT group.
Minor comments: 1) Figure 1b: The authors should add another sub-section on the lower end of y-axis, as the current graphical illustration gives a rather confusing perspective that the vector and LDLRAD3 groups do not generate any viral titer whereas the actual scenario is that the vector has a 100% relative viral RNA level.
2) Figure legend for Figure 6: PDB IDs have to be in uppercase.
3) There are a few typing errors across the manuscript

Reviewer #1 (Remarks to the Author)
Major: 1.The data in Figure 1d-k were generated with lentivirus pseudotypes.These lentivirus pseudotypes are unlikely to contain properly assembled E2/E1 protomers (with icosahedral assembly).So, while this experiment is well controlled (e.g., expected phenotypes are seen with Mxra8 overexpression with chikungunya virus pseudotypes), it remains critical to also use a different system that faithfully recapitulates alphavirus spike protein organization to make a claim on receptor use for SFV, BEBV, and RRV (e.g., chimeric Sindbis viruses, which are the standard in the field, or authentic alphaviruses).
For example, a separate study found only low levels of entry of alphavirus replicon-based SFV reporter virus particles with cells overexpressing LDLR; but a much more significant effect was seen with VLDLR overexpression (PMID: 34929721).Structural analysis of VLDLR bound to SFV virus-like particles also suggests that there may be important requirements for receptor assembly near icosahedral axes of the E2-E1 spike proteins (PMID: 37098345).It is thus possible that sites that might be exposed for receptor binding by LDLR on SFV-lentivirus pseudotypes might not be exposed on properly assembled E2-E1 spikes in the context of native or native-like virions.Response: Authors thank the reviewer for the comment and suggestion.We understand the concern for the organization of E2-E1 on HIV pseudo-viruses, which might affect the interaction with LDLR.However, we would like to point out that our preliminary model of this interaction does not rely on the organization of E2-E1 on virus particles (Fig. 6e of the main text).Nevertheless, we have now constructed recombinant reporter viruses of rSFV-mCherry, rRRV-mCherry, and rSINV-BEBV-mCherry (chimeric Sindbis viruses).Binding and internalization experiments indicated that all three recombinant viruses had increased infection in the presence of LDLR overexpression, in a similar way to our previous results with HIV pseudo-viruses.Please see the Extended Data Fig. 6.In addition, the extent of each recombinant reporter viruses benefiting from LDLR followed the trend displayed in HIV pseudo-virus experiments (Fig. 1c-k of the main text).For example, RRV benefits less from overexpression of LDLR than SFV, GETV and BEBV in both assays.These new data reinforced our initial conclusion with HIV pseudo-viruses.The construction and validation of these recombinant viruses were put into Extended Data Fig. 1.
2. Critical assays for SFV, BEBV, and RRV are missing to conclude that LDLR is a receptor for these viruses.These assays include: Extended Data Fig. 6 (a) Showing that LDLR binds to the E2-E1 proteins of these viruses through immunoprecipitation experiments, ELISAs, or BLI experiments.
(b) Assays showing that LDLR expression results in binding to cells and virus internalization.
(c) As noted above, assays with authentic viruses are missing.Response: Thank you for the suggestions.In the response above, we have provided new data of binding and internalization experiments using recombinant viruses of rSFV-mCherry, rRRV-mCherry, and rSINV-BEBV-mCherry.In addition, we have now included the data from immunoprecipitation experiments with HA-tagged envelope proteins of SFV, BEBV, and RRV.As shown below, GST-LBD protein can interact with p62-E1-HA of SFV, BEBV, and RRV.The new IP data is added to Extended Data Fig. 9a-c.
3. VLDLR, a known alphavirus receptor that is very closely related to LDLR and is also a receptor for multiple alphaviruses including SFV, was not tested anywhere in the manuscript.VLDLR (and ApoER2) are two canonical members of the LDLR family that have similar structural organization and sequence homology in their ligand-binding domains to LDLR (while LDLRAD3, which the authors tested, is quite different).While the manuscript centers on LDLR, interest would be significantly increased if the authors demonstrated that LDLR but not VLDLR or ApoER2 are cellular receptors for GETV.This finding would have important implications to the general understanding of the evolution of alphavirus receptor specificities.
In a related question, were VLDLR and ApoER2 on the list of the 150 membrane proteins tested in their screen?The list of membrane proteins should be provided in the supplemental materials for completeness.

Response:
Authors thank the reviewer for this good suggestion.We have tested the effects of VLDLR and ApoER2 (LRP8) on GETV.As shown below, indeed, VLDLR and ApoER2 can also significantly promote GETV infection in HEK 293T.In addition, to analyze whether LDLR can act independently as receptor we overexpressed LDLR in K562 cells lacking VLDLR and LRP8 (DOI: 10.1038/s41586-021-04326-0), and found that LDLR could significantly promoted viral infection.This suggests that these three closely related receptors, which belong to the LDLR receptor family Extended Data Fig. 9 and contain CR repeats, can all act as receptors of one and the same alphavirus.Interestingly, according to previous research, VLDLR CR3 binds SFV E1-DIII (DOI: 10.1016/j.cell.2023.03.032), while our data shows that for LDLR CR4, CR5 can also mediate GETV infection.There seems to be a general rule for alpha viruses and LDLR receptor family.However, it requires much more work to substantialize this theory and extend it to all representative alpha viruses.In our opinion, comprehensive analysis of the relationships between LDLR receptor family proteins and alphaviruses falls beyond the scope of this study.For this reason, we decided not to include the (compared to the extensive analysis performed with LDLR) rather limited data of VLDLR and LRP8 to the manuscript.Nevertheless, it represents an attractive topic for future studies.
In addition, it should be noted that VLDLR and ApoER2 were not in the list of the 150 membrane proteins analyzed in this study; so the fact that they were not initially discovered as receptors for GETV is due to the limited size of our library.The list of membrane protein present in the library has now been provided in the Extended Data Table 1. 4. Biolayer interferometry assays in Figure 3i.The authors dipped sensor tips that were coated with receptors into solution containing VLPs, and report measuring an affinity of 263 nM.There would be substantial avidity in the system, which would falsely decrease the off rate.Any KD determination would therefore be uninterpretable.For affinity measurements, the virus would have to be immobilized on the sensor tip and the receptor kept in solution to minimize avidity.

Response:
Authors agree that coating the sensor with VLPs will have less avidity issue.However, we do want to point out that there are also many publications that did the experiments in the reverse way (DOI: 6.For the confocal microscopy studies (Figure 2k), how many images were used to quantify binding?
Could additional images be provided in the supplement? Response: We apologize for lack of details in the image analysis.As suggested by other reviewers, we re-did the experiment with a higher dose of the virus (MOI=200).This new data is now displayed in Fig. 2b.For image analysis, we quantified the number of virions in each experimental group using three individual images taken with full field-of-view (as shown below).ImageJ software was set to randomly pick up 75 cells for quantification in each experimental group.And a representative zoomin image was put into the figure for illustration purpose.We will provide all the full field-of-view images used for quantification in a raw data file to the manuscript.
Extended Data Fig. 8d 7. Abstract: "Although several receptors have been identified for alphavirus entry, they could not explain broad host range and multi-tissue tropism of alphaviruses, indicating the existence of additional receptors."This reviewer does not agree with this statement.Another, very closely related LDLR-related family receptor, VLDLR, has already been described as a conserved receptor for SFV, and orthologs from vertebrates and invertebrates are functional receptors.Would recommend rephrasing to say that some of the previously identified receptors cannot explain the broad host range and tissue tropism of certain alphaviruses.

Response:
Thanks for pointing this out.We have changed the sentence to " So far several receptors have been identified for alphavirus entry, however, they cannot explain the broad host range and tissue tropism of certain alphaviruses, such as Getah virus (GETV), indicating the existence of additional receptors."Please see lines 21-24.

Minor:
Lines 70-71: "However, ApoER2 and VLDLR are almost exclusively expressed in the central nervous system" While ApoER2 expression is mostly limited to the brain, VLDLR is more widely expressed, with expression in the heart, muscle, lung, kidneys, and adipose tissue.See reference PMID: 21554715.Response: Thanks for pointing this out.We have changed the sentence (lines 77-81) to "Similarly, for SFV, ApoER2 is almost exclusively expressed in the central nervous system.Although VLDLR is expressed in multiple tissues, it has been observed that the absence of VLDLR or presence of antibodies against VLDLR do not completely block the infection of SFV in Vero, U20S, A549, Huh7 and other cells".1.The paper suffers from a serious deficiency of careful documentation of the various constructs used.The library of 150 membrane proteins are tagged with eGFP-there is no documentation of whether the tag is on the N-or C-terminus, and whether it affects delivery of the protein to the membrane.

Response:
We sincerely apologize for the lack of detailed information of the constructs used in this study.First of all, to make it clear, plasmids in the library of membrane proteins were constructed to have EGFP as a co-expressing gene, instead of a fusion-tag.Please see the plasmid map below.The coexpression of EGFP as a separate protein in theory does not affect the localization of the membrane proteins in the library.We amended the Methods with the details of plasmid construction (lines 416-419).Nevertheless, we further confirmed the localization of the membrane protein hits in our study by laser scanning confocal microscopy (TIMD4 and LDLR).
More importantly, for the N-tagged LDLR constructs, there is no information on whether the FLAG tag is placed after the signal sequence, and if not, how the protein would be inserted into the ER.
Response: Actually, the constructs for LDLR proteins were designed to have Flag tag in the C terminus, which was described in lines 449-453 of the Methods but was put in a wrong location in Fig. 3a of our last draft.We sincerely apologize for the confusion caused by our carelessness.We have now corrected Fig. 3a to reflect the correct location of the Flag tag in our revised manuscript.
The studies lean heavily on the use of a soluble LDL LBD-GST protein, which is used to block infection in cell culture and in mouse studies, and to produce an antibody that is used in cell blocking experiments.This LBD-GST protein is produced in bacteria.There is no documentation that this heavily disulfide-bonded protein is folded correctly, or references to puvlpshed work that might support this.

Response:
First of all, it has been demonstrated that soluble E.coli expressed GST-CR domains can functionally block VSV infection (doi: 10.1038/s41467-018-03432-4). In addition, for further proof, we have now provided comparative analysis of E.coli expressed GST-LBD with a mammalian cell (Expi293F) expressed LBD-Fc in blocking virus infection.As shown below, GST-LBD and LBD-Fc block GETV infection to a similar extent, which confirms our E.coli expressed GST-LBD is functional.We have added this to Extended Data Fig. 10.

LDLR TIMD4
Might this explain why the western blot and flow cytometry studies use commercial Abs to LDLR or FLAG, rather than the polyclonal antibody they generated?Response: Commercial antibodies were used to in western blot and flow cytometry simply because these experiments were performed before our own anti-LDLR were available.
2. It is critical to confirm that the various LDLR mutants are expressed on the cell surface.This is addressed by flow cytometry and receptor staining.The receptors are described to be N-terminally tagged i.e. the FLAG tag should be localized extracellularly.However, the flow cytometry methods state that cells were permeabilised to detect the FLAG tag.From this they then conclude the receptor cell surface levels.This is not accurate as permeabilisation will allow the entire receptor population to be labelled (i.e both cytoplasmic and PM localised).Without cell surface confirmation of all receptor constructs, the data are not conclusive.(The methods also read like cells are fixed prior to trypsinisation, which would prevent the cells from coming off the plate for the flow analysis).

Response:
We thank author for the comment and apologize again for the confusion caused by grammatical errors and inaccurate description.As mentioned above, the constructs for LDLR proteins were in fact designed to have Flag tag at the C terminus.That was the reason why we did permeation before staining the cells.We have now used confocal microscopy to confirm the membrane localization of overexpressed LDLR mutants and added this data to Extended Data Fig. 4b.b a Extended Data Fig. 10 Extended Data Fig. 4b E1 and E2.On the other hand, VLDLR binds to the DⅢ domain of the SFV E1 protein close to the envelope membrane and does not interact with the E2 of SFV.".Please see lines 69-73. 5. Line 71 is inaccurate.Both NCBI and Human protein atlas databases report very wide tissue expression of at least human VLDLR.Response: We thank reviewer for pointing this out.In the revised manuscript, we have changed the sentence to "Similarly, for SFV, ApoER2 is almost exclusively expressed in the central nervous system.Although VLDLR is expressed in multiple tissues, it has been observed that the absence of VLDLR or presence of antibodies against VLDLR do not completely block the infection of SFV in Vero, U20S, A549, Huh7 and other cells".Please see lines 77-81.
6. Please adjust text in introduction to reflect that the Clark et al paper tested VLDLR from many species including mosquito.

Response:
We thank reviewer for pointing this out.In the revised manuscript, we have changed the sentence (lines 65-69) to " In addition, very low-density lipoprotein receptor (VLDLR) from many species (including mosquitoes) and apolipoprotein E receptor 2 (ApoER2) have recently been identified as viral receptors in vertebrate and invertebrate cells for SFV, EEEV and SINV.".We have not analyzed the difference between those two bands of LDLR specifically.Because LDLR is N-glycosylated, our speculation is that the lower band is a form of LDLR with less degree of Nglycosylation, as suggested in literature (DOI: 10.1074/jbc.M113.545053).

Reviewer #3 (Remarks to the Author)
In this study, the authors identified LDLR as a crucial receptor involved in entry of several viruses belonging to the Alphavirus genus into target cells, whereby Getah virus was the main focus of the study due to biological safety reasons.The overall experimental strategy was well-designed.However, there is major concern if LDLR is indeed the crucial receptor for these viruses without validation experiments with in-vivo knockout model of LDLR.In-vivo model of LDLR knockout are well established and has been utilized for diseases such as hypercholesterolemia.

Major points:
1) The authors have constructed an expression plasmid library for 150 mouse membrane proteins, in order to identify the specific protein(s) which positively affect alphavirus infections.What are the selection criteria of these proteins, have these proteins been shown in the past to be associated with the infection capacity of other viruses? Response: We acknowledge that the library used in this study is of limited size.It was obtained as part of our effort to build a library of all the murine membrane proteins (about 10,026 proteins in total; the completion of this project may take a decade).By the time this study was initiated we had validated the expression constructs for the first 150 membrane proteins (Extended Data Table 1).Initially, we did not know whether or not they are related to the virus infection.Aside of our main findings, this study represents a proof that construction and use of such libraries is a valid approach.We believe that in our larger library, which is still under construction, we will find more and more unknown host factors that can affect the alphavirus life cycle.However, since our identification of LDLR from this limited size library is kind of serendipity, we decided to move this result to Extended Data Fig. 2b to avoid misleading the audience.
2)It will be useful for the authors to provide a list of the tested proteins including those which were negative hits in this study, as it can serve as an important source of information for future works by the readers.

Response:
A list of the 150 membrane proteins is now presented as the Extended Data Table 1.
3)Figure 2j: Can the authors provide a clear description on the y-axis of the graph?Is the y-axis presented as relative ratio of internalization:binding data?The error bars are too huge in my opinion to deduce that there is an effect on the virus internalization, while there are some data points which showed similar levels of vRNA as the WT.

Response:
Thank you for the comment.We apologize for the confusion.Our initial intention was to normalize the amount of internalized virions to the amount of bound.We have now realized that this is unnecessary as is shown by literatures (DOI: 10.1038/s41586-018-0121-3;). We have therefore removed this ratio and combined the data of the bound and internalized in the same way as in the literatures (Fig. 2a).We also went back to check the reason for the unusually large error bars.The consistency of the qPCR is significantly improved after we switch to a different qPCR kit.The new data is packed into Fig.2a in the revised manuscript.4)In Figure 2t, the authors showed that JEV is among the virus which are not affected by LDLR deficiency in the host cells.However, this is in contrast to a previous finding (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238074/) which mechanistically demonstrated that LDLR is an important host factor required for JEV entry.The authors will need to address this contrasting data.

Response:
We thank reviewer for pointing this out.This is a very interesting finding.The apparent contradiction could be the result of different cell lines used in the study.We used BHK-21 cell, which is a baby hamster kidney fibroblast cell, while the literature referred by reviewer used A549, a human lung cancer cell line.It is possible that the dependency of JEV on LDLR as receptor is different in these two cell lines.JEV might have an alternative, highly expressed receptor in BHK-21 cells, making it insensitive the deletion of LDLR.Since JEV is not in the focus of this study, we did not perform further experiments to test our hypothesis and removed results with JEV from the manuscript.5)Although the mouse sera used in this study was collected from mice subcutaneously injected with GST-LBD, how did the author confirm the presence of anti-LDLR antibodies in the sera generated from their mouse model?Was there any neutralization test being performed using the generated sera?
The authors need to convincingly show that the inhibition observed on the rGETV-EGFP infection within the different cell lines tested are indeed due to the presence of anti-LDLR antibodies.
Response: Thanks for your suggestion.We have now included ELISA assay of the anti-LDLR mouse sera against purified GST-LBD in Extended Data Table 2. Further, we performed antibody validation and cross-reactivity experiments.As shown in the figure below, the LDLR antibody we used for neutralization studies could react with GST-LBD and full-length LDLR-Flag expressed in HEK 293T cells, but not with VLDLR-Flag.The result indicated that our antibody could specifically recognize LDLR but not its highly related family member VLDLR.We included the new data in Extended Data Fig. 11.

Response:
Thanks for the suggestion.We agree that in vivo studies in a LDLR KO mouse would be helpful for evaluating the importance of LDLR in the GETV-caused pathology.However, we think the focus of our current study is the discovery of LDLR as a GETV receptor.In addition, the in vivo experiments require LDLR-KO suckling mice, which we do not currently have.Therefore, we have to leave this to our next manuscript.8)Figure 6b and c: It is inconclusive for the authors to deduce that the CR4 region are also essential for promoting GETV infection as there were still statistically significant upregulation of the EGFP+ cells and viral titers in the absence of CR4.Line 223: Was the promoting effect in the absence of CR4 statistically compared to CR5?It is inaccurate for the authors to state that "deletion of CR4 statistically reduced the promoting effect of LDLR expression", as the statistics performed were on the upregulation of EGFP+ cells and viral titers relative to the WT group.Extended Data Fig. 11 Response: Thank you for pointing this for us.Indeed, this is due to lack of statistics analysis in the figure .We have now amended Fig. 6b and 6c with statistics analysis of the negative impact of EGFP+ cells and viral titers of CR4 and CR5 mutants relative to the full-length LDLR.We further modified the text (lines 285-290) as follows: "After deletion of CR4, EGFP+ cells and viral titers were still significantly upregulated compared with those in the group without overexpression of LDLR protein, but to a much lesser extent compared with those overexpressing full-length LDLR protein (Fig. 6bd).This suggests that both CR4 and CR5 are essential for the infection of GETV, with CR5 playing the most dominant role".

Minor comments:
1) Figure 1b: The authors should add another sub-section on the lower end of y-axis, as the current graphical illustration gives a rather confusing perspective that the vector and LDLRAD3 groups do not generate any viral titer whereas the actual scenario is that the vector has a 100% relative viral RNA level. Response:

Figure 1 :
Figure1: HEK293T cells are used for overexpression studies, but how much native expression of LDLR is there on these cells?
Fig 8, the comparison should include non-permissive host LDLR, and the mosquito homologue.6. Fig 2n presents the rather surprising finding that the deletion of MXRA8, which decreases GETV infection, can be rescued by expression of LDLR.What about the converse experiment---can the LDLR deletion or the LDLR/MXRA8 double deletion be rescued by MXRA8?The result currently is presented without any discussion of what it might mean.
7. It's very hard to see plaques in ED Fig 1e.Please fix figure.8. Why does LDLR-FLAG show two bands in ED Fig 1g? Reviewer #3 (Remarks to the Author): .1128/JVI.01871-16DOI:10.1007/s00216-015-8735-x).That being said, in order to completely rule out the avidity issue, we replaced VLP with purified p62-E1 protein, and re-did BLI experiment.As shown in the figure below, we recorded a KD value of 273 nM in this way.We added this new data to Extended Data Fig.8d.5.Antibody blocking assays in Figure4e-h rely on mouse anti-LDLR serum, which may have some cross-reactivity with other LDLR family members (e.g., with VLDLR) and confound the results of this assay.Could the authors comment on how this antibody was validated, and is it possible that it may cross-react with VLDLR, LRP1, etc.?Response: Thanks for your suggestion, we have now included validation data of anti-LDLR serum.Briefly, it is found that this polyclonal anti-LDLR serum recognize GST-LBD and LDLR-Flag expressed in HEK 293T cells but do not recognize VLDLR-Flag.This new data is now included in Extended Data Fig.11of the revised manuscript.

Figure 1 :
Figure 1: HEK293T cells are used for overexpression studies, but how much native expression of LDLR is there on these cells?Response: Please refer to the Western Blots shown below for the level of native expression of LDLR of HEK 293T in comparison to overexpression of Flag-tagged LDLR.
7. It's very hard to see plaques in ED Fig 1e.Please fix figure.Response: We have replaced it with higher resolution image and due to other revisions it is now in Extended Data Fig. 1e.8. Why does LDLR-FLAG show two bands in ED Fig 1g? Response: